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Depression of the Glass Transition Temperature of Sucrose Confined in a Phospholipid Mesophase Evgenyi Y. Shalaev*,†,‡ and Peter L. Steponkus† Department of Crop and Soil Sciences, Cornell University, Ithaca, New York 14853, and Groton Laboratories, Pfizer Inc., Groton, Connecticut 06340 Received April 26, 2001. In Final Form: June 8, 2001 The glass transition of sucrose in 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)/sucrose mixtures was studied by differential scanning calorimetry. Mole ratios of 1:2, 1:1, and 2:1 DOPE/sucrose mixtures were dehydrated at osmotic pressures ranging from 41 to 311 MPa through vapor-phase equilibration. Two glass transitions were observed in 1:2 DOPE/sucrose mixtures that were dehydrated at osmotic pressures >81 MPa. The first glass transition temperature (Tg) was lower than that of pure sucrose solutions that were dehydrated under identical conditions. The second Tg of the 1:2 DOPE/sucrose mixture was similar to the Tg of sucrose solutions that were dehydrated in the absence of DOPE. In the 2:1 and 1:1 DOPE/sucrose mixtures, only one Tg was observed at a temperature lower than that of pure sucrose. We propose that depression of the Tg of sucrose in the DOPE/sucrose mixtures is the result of a confinement effect that occurs in the sucrose solution that is sequestered in the aqueous cores of the hexagonal II phase of DOPE with a radius of ∼2 nm.
Introduction Aqueous solutions of carbohydrates commonly form a noncrystalline solid (glassy) state during freezing or desiccation.1 The ability of sugars to form the glassy state is considered to be one of the major mechanisms of stabilization of liposomes and biological membranes by sugars against stresses imposed by freezing and desiccation.2 In particular, transformation of a sugar matrix from a liquid to a glassy state has a major impact on phase behavior of phospholipids. Koster et al. observed that the lamellar liquid crystal (LR)-to-gel (Lβ) phase transition temperature (Tm) of phosphatidylcholine (PC) species dehydrated in the presence of sugars decreased significantly when the glass transition temperature, Tg, of sugar was greater than the Tm of the lipid.3 In a more detailed study, Zhang and Steponkus4 demonstrated that the relationship between the glass transition of a sugar and depression of the Tm of a PC species is more complex. Depression of the Tm only occurred if the phospholipid was in the LR phase when the sugar matrix underwent a glass transformation.4 This finding has recently been confirmed by Koster et al.5 Zhang and Steponkus suggested4 that depression of the Tm of a PC species is caused by rigidity of the sugar glass rather than a direct lipid/ sugar interaction;6 the lipid phase transition is accompanied by area changes, and the rigid sugar matrix resists the area changes that might create an increase in lateral pressure. For another major class of phospholipids, phosphatidylethanolamine (PE), it was observed that the * To whom correspondence may be addressed at Groton Laboratories, MS 8156-04, Pfizer Inc., Eastern Point Rd, Groton, CT 06340. † Cornell University. ‡ Pfizer Inc. (1) Franks, F. Pure Appl. Chem. 1997, 69, 915. (2) Koster, K. L. Plant Physiol. 1991, 96, 302. (3) Koster, K. L.; Webb, M. S.; Bryant, G.; Lynch, D. V. Biochim. Biophys. Acta 1994, 1193, 143. (4) Zhang, J.; Steponkus, P. L. Cryobiology 1996, 33, 624. (5) Koster, K. L.; Lei, Y. P.; Anderson, M.; Martin, S.; Bryant, G. Biophys. J. 2000, 78, 1932. (6) Crowe, J. W.; Hoekstra, F. A.; Nguyen, K. H. N.; Crowe, L. M. Biochim. Biophys. Acta 1996, 1280, 187.
thermotropic hexagonal II (HII)-to-Lβ phase transition was hindered, and PE was trapped in metastable HII phase when the viscosity of sucrose phase approached 107-1010 Pa s.7 In the PE/sucrose mixtures, the effect of sucrose on the HII-to-Lβ phase transition is believed to be a kinetic effect rather than a thermodynamic effect. Despite the fact that the effect of a sugar glass transformation on the phase behavior of phospholipids is well documented, the reverse effect, i.e., the effect of a phospholipid on the Tg of sugar, has been less studied. One of the first observations of the effect of a phospholipid on the glass transition of a sugar was reported by Shalaev and Steponkus7 where two glass transitions of sucrose were detected in 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE)/sucrose mixtures. The two glass transitions were explained on the basis of two compartments of sucrose molecules. The present communication explores this observation in more detail. In particular, we suggest that DOPE affects the Tg of sucrose through a “confinement” effect8-10 when sucrose molecules are confined within cylinders of the HII phase of DOPE with a radius of several nanometers. Materials and Methods Sucrose was purchased from Fisher Scientific as an ACS reagent. 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) was purchased from Avanti Polar Lipids as a chloroform solution and used without further purification. DOPE/sucrose 2:1, 1:1, and 1:2 mixtures (mole ratio) were prepared by mixing a solution of sucrose and a suspension of DOPE in water followed by 10 freeze-thaw cycles with vortexing between the cycles. The DOPE/sucrose mixtures were dehydrated through vapor phase equilibration over saturated salt solutions at 0 and 30 °C at osmotic pressures ranging from 40 to 305 MPa (relative humidi(7) Shalaev, E. Y.; Steponkus, P. L. Biochim. Biophys. Acta 2001, in press. (8) Keddie, J. L.; Jones, R. A. L.; Cory, R. A. Europhys. Lett. 1994, 2759. (9) Jackson, C. L.; McKenna, G. B. J. Non-Cryst. Solids 1991, 131133, 221. (10) Forrest, J. A.; Dalnoki-Veress, K.; Stevens, J. R.; Dutcher, J. R. Phys. Rev. Lett. 1996, 77, 2002.
10.1021/la010617+ CCC: $20.00 © 2001 American Chemical Society Published on Web 07/26/2001
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ties from 75 to 11%). Osmotic pressures, Π, for those solutions were calculated as11,17
Π ) -(RT/Vw) ln(RH/100)
(1)
where R ) 8.31 J mol-1 K-1 is the universal gas constant, T is the temperature in K, Vw ) 18 × 10-6 m3 is the partial molar volume of pure water, and RH is the relative humidity of the saturated salt solution. Differential scanning calorimetry (DSC) experiments were performed with a Perkin-Elmer DSC-7 instrument. An empty aluminum pan was used as a reference. The instrument was calibrated using melting points of water and indium. Heating and cooling rates were 10 and 40 °C/min; the Tg measured at 40 °C/min was ∼2 °C higher than that measured at 10 °C/min. Samples were cooled to e-120 °C followed by immediate heating to g30 °C; cooling-heating cycles were repeated several times. The glass transition temperatures are reported as the onset temperatures. Water content was determined gravimetrically after DSC and X-ray diffraction experiments.12 Other experimental details for sample preparation, water content analysis, and DSC measurements are described elsewhere.7,12,13 X-ray diffraction powder patterns were obtained for the 1:1 and 1:2 DOPE/sucrose mixtures dehydrated at 163 MPa (water content 1.4 and 2.5 wt %, respectively) at the Cornell High Energy Synchrotron Source. A wide-angle diffuse ring and one smallangle ring were observed (the patterns are not shown). d spacings were 0.45-0.47 nm for the wide-angle diffuse diffraction ring and 4.7 nm for the small-angle ring in both 1:2 and 1:1 DOPE/ sucrose mixtures at 30 °C. The phase state of DOPE in these mixtures was tentatively identified as the HII phase;7 lack of higher-order small-angle diffractions did not allow us to unambiguously determine the phase. Dimension of the cylinders of the HII phase in the 1:1 mixture at 1.4 wt % water, Rnl2, was calculated to be 1.85 nm using Luzzati’s method14,15
Rnl2 ) (2dhex2(1 - φl))/(π31/2)
(2)
where Rnl2 is radius of the cylinder, dhex is d spacing of the HII phase, φl ) 1/(1 + (1 - c)νs/cνl ) is the volume fraction of lipid fraction of the sample, νs and νl are partial specific volumes of sucrose and phospholipid, respectively, and c is weight fraction of lipid. νl was taken as 1 g/cm3,16 and density of a nonlipid (sugar) portion was assumed to be equal to 1.5 g/cm3.17,18
Results Representative DSC heating curves of 1:1 and 1:2 DOPE/sucrose mixtures and a sucrose solution that were dehydrated at 81 MPa at 30 °C are shown in Figure 1a. Only one glass transition of sucrose was observed in the 1:1 DOPE/sucrose mixture and the pure sucrose solution, whereas two glass transitions were observed in the 1:2 mixture. Thermotropic phase transitions of DOPE were not observed in the DOPE/sucrose mixtures dehydrated at g81 MPa.7 The temperature of the second glass transition (Tg2 ) -16 °C) in the 1:2 DOPE/sucrose mixture was close to the Tg of the sucrose solution (Tg ) -18 °C), (11) Glasstone, S. Textbook of physical chemistry; D. Van Nostrad Co., Inc.: Princeton, NJ, 1946; pp 669-670. (12) Shalaev, E. Y.; Steponkus, P. L. Thermochim. Acta 2000, 345, 141. (13) Shalaev, E. Y.; Steponkus, P. L. Biochim. Biophys. Acta 1999, 1419, 229. (14) Luzzati, V. In Biological Membranes; Chapman, D., Ed.; Academic Press: London & New York, 1968; p 71. (15) Rand, R. P.; Fuller, N. L.; Gruner, S. M.; Parsegain, V. A. Biochemistry 1990, 29, 76. (16) Rand, R. P.; Parsegain, V. A. Biochim. Biophys. Acta 1989, 988, 351. (17) Plato, Wiss. Abh. Kaisel., Normal Aichung Kommission, SpringerVerlag: Berlin, 1901. Citation from: Reiser, P.; Birch, G. G.; Mathlouthi, M. In Sucrose. Properties and Applications; Mathlouthi, M., Ed.; Blackie Academic&Professional: London, 1995; p 190. (18) Shamblin, S. L.; Huang, E. Y.; Zografi, G. J. Therm. Anal. 1996, 47, 1567.
Figure 1. (top) Representative DSC heating curves of 1:2 and 1:1 DOPE/sucrose mixtures and pure sucrose dehydrated at 81 MPa at 30 °C. Two sucrose glass transitions were observed for DOPE/sucrose ) 1:2 mixtures but only one Tg for pure sucrose and 1:1 mixture. (bottom) Representative DSC heating curves of the DOPE/sucrose ) 1:2 mixtures dehydrated at 81 MPa at 30 °C and annealed at two different temperatures. Annealing time was 30 min. Tg - Ta ≈ 5 °C. Solid curves represent annealed samples. Broken curves represent second heating.
whereas the low-temperature glass transition (Tg1 ) -31 °C) was similar to that of the 1:1 mixture (Tg ) -36 °C) and lower than that of pure sucrose. Annealing experiments were performed to verify that both thermal events (Tg1 and Tg2) were the result of a glass transition. A 1:2 DOPE/sucrose mixture dehydrated at 81 MPa was annealed at two different temperatures, Ta, for different time periods. The Ta values were chosen to be -35 and -21 °C, which were ∼5 °C lower than Tg1 and Tg2, respectively. DSC heating curves after annealing at -35 and -21 °C for 30 min are shown in Figure 1b as examples. Solid curves are given for annealed samples (first heating scan), and broken curves are for the second heating scans. A thermal overshoot (enthalpy recovery) was observed after annealing at both -35 and -21 °C. Enthalpy recovery is a typical phenomenon observed when glassy materials are annealed below their Tg.19 Hence, the results of the annealing experiments support the assignment of both endothermic steps to a glass transition. Two glass transitions of sucrosewere observed in 1:2 DOPE/sucrose mixtures dehydrated at osmotic pressures ranging from 81 to 311 MPa at both 0 and 30 °C, but only one glass transition was detected in 1:1 and 2:1 mixtures (DSC scans are not shown). The Tg values of sucrose in DOPE/sucrose mixtures and in a pure sucrose solution as a function of osmotic pressure are given in Figure 2. The results for samples dehydrated at 0 and 30 °C are shown in separate graphs for the sake of clarity. The Tg of sucrose in the 1:1 and 2:1 DOPE/sucrose mixtures was lower than the Tg of pure sucrose. The Tg2 values in the 1:2 mixtures (19) Hodge, I. M. J. Non-Cryst. Solids 1994, 169, 211-266.
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Figure 2. Tg vs osmotic pressure of the DOPE/sucrose mixtures dehydrated at 0 (top) and 30 °C (bottom): O and broken line, DOPE/sucrose ) 1:2, Tg1; 0 and dotted thick line, DOPE/sucrose ) 1:2, Tg2; 4 and dot-and-dash line, DOPE/sucrose ) 1:1; ] and dotted line, DOPE/sucrose ) 2:1; 9 and solid line, sucrose at 30 °C. Lines are given as a visual aid.
Figure 3. ∆Tg ) Tg(DOPE/sucrose) - Tg(sucrose) vs osmotic pressure. Tg1 values only are plotted for 1:2 DOPE/sucrose mixtures. O, b, 1:2 DOPE/sucrose mixtures dehydrated at 0 and 30 °C, respectively; 0, 9, 1:1 DOPE/sucrose mixtures dehydrated at 0 and 30 °C; 4, 2:1 DOPE/sucrose mixtures dehydrated at 0 °C. Lines are given as a visual aid.
were similar or slightly higher than the Tg of the pure sucrose solution equilibrated at corresponding osmotic pressures (Figure 2) whereas the Tg1 values were similar to the Tg in DOPE-rich mixtures (1:1 and 2:1) and significantly lower than that of the pure sucrose solution. The difference in the Tg between sucrose and DOPE/ sucrose mixtures, ∆Tg, increased with the osmotic pressure as shown in Figure 3. Discussion The data suggest that there are two sucrose compartments in 1:2 DOPE/sucrose mixtures dehydrated at osmotic pressures g81 MPa: a “bulk” compartment with a Tg that is similar to the Tg of pure sucrose dehydrated at corresponding osmotic pressures, and a compartment in which Tg is lowered by the presence of DOPE molecules. DOPE-rich mixtures (2:1 and 1:1 DOPE/sucrose) contain the second compartment (the one with a Tg that is lower than the Tg of the sucrose solution) only. Furthermore, we suggest that these two compartments represent sucrose molecules within the HII cylinders of DOPE and bulk sugar phase as presented schematically in Figure 4. In DOPErich mixtures, all sucrose molecules are located within cylinders of the HII phase of DOPE, i.e., the bulk sugar phase is absent. It is known that the Tg of glass formers that are confined in small pores or in thin layers can be different from the Tg of the same material in the bulk solution (see refs 8-10 for example). The Tg in a small system can be either higher or lower than the Tg in the bulk depending on the balance between the effect of a free surface and the effect of the support (substrate). It has been suggested that the effect of a free surface is to decrease the Tg whereas the effect of substrate is to increase the Tg.10 A depression of the Tg occurred when a size of a system is comparable with the size of a cooperative unit (or, in other words, with characteristic length of glass transition) whereas increase
Figure 4. A “drawing” of the HII phase in sucrose-rich DOPE/ sucrose mixtures. “S” symbol represents sucrose molecules. Water molecules are not shown. Tg1 corresponds to the glass transition of sucrose confined in the HII phase of DOPE, and Tg2 corresponds to the glass transition of a bulk sucrose phase. The figure was adapted from Kozlov et al. with modifications. (Kozlov, M. M.; Leikin, S.; Rand, R. P. Biophys. J. 1994, 67, 1603-1611.)
in Tg can be explained as the result of interaction (for example, hydrogen bonding) of a glass former with the support.10 The confinement effect on the Tg was observed when the pore size or the layer thickness was on a scale of several to tens of nanometers, which is a typical size of the aqueous portion of cylinders of the HII phase. For example, radius of the cylinders of the HII phase, which confines sucrose molecules, was estimated to be Rnl ) 1.85 nm for the 1:1 DOPE/sucrose mixture dehydrated at 163 MPa. A significant decrease in the Tg of sucrose confined in DOPE mesophase (Figure 3) suggests that a free surface has a major impact on properties of sucrose confined in the HII cylinders whereas possible contribution from interaction between sucrose molecules and DOPE (sup-
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Figure 5. Comparison of the experimental and calculated water contents for 1:2 and 1:1 DOPE/sucrose mixtures: O, b, water contents obtained in desorption experiments at 30 °C for 1:1 and 1:2 DOPE/sucrose mixtures, respectively; 0, 9, water contents obtained in desorption experiments at 0 °C for 1:1 and 1:2 DOPE/sucrose mixtures, respectively. Lines represent calculated water contents assuming that DOPE and sucrose hydrated independently of each other for 1:1 (solid line) and 1:2 (broken line) DOPE/sucrose mixtures. Hydration data which were used to calculate theoretical hydration of DOPE/sucrose mixtures were from ref 7 for sucrose (up to osmotic pressure 157 MPa) and for DOPE in the HII phase at 30 °C, and from (Saleki-Gerhardt, A.; Zografi, G. Pharm. Res. 1994, 8, 11661173) for sucrose at osmotic pressure 305 MPa (corresponds to relative humidity 11%).
port) is not significant. This is different from PC/sugar mixtures where the hydrogen bonding between OH groups
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of carbohydrate and phosphate groups of the phospholipid was proposed based on FT-IR results.20 It is possible that PC and PE species, which had different phosphate headgroups, have a different ability for hydrogen bonding with sugars. Another possibility is that the difference in the interaction of sugars with DOPE and DPPC is due to difference in the phase state of phospholipids. DOPE was in the inverted hexagonal phase whereas DPPC was in the lamellar phase. It should be noted that an alternative explanation on impact of DOPE on Tg of sucrose can be suggested. A water content in the bulk solute phase may be different than that in the confined phase at the same water activity,21 and the Tg depression may be a result of a higher water content in the confined sucrose phase. However, data on water sorption of DOPE/sucrose mixtures do not support this suggestion. Water contents of DOPE/sucrose mixtures were similar to or lower than the theoretical hydration which was calculated assuming that sucrose and the HII phase of DOPE in the DOPE/sucrose mixtures dehydrate independently of each other (Figure 5 and ref 7). Hence, we conclude that depression of the Tg of sucrose confined in the HII phase is a result of a free surface effect when dimension of the HII cylinders (radius of ∼2 nm) is comparable with a characteristic length of a glass transition of sucrose near Tg. Note that similar characteristic lengths of glass transition (ranged from 1 to 3.5 nm) were reported for a number of glass-forming materials.22 Acknowledgment. This work was supported by a grant (DE-FG02-84ER13214) from the United States Department of Energy. We wish to thank Dr. Ernest Fontes for his expert assistance with the X-ray diffraction experiments. LA010617+ (20) Crowe, L. M.; Crowe, J. H.; Chapman, D. Arch. Biochem. Biophys. 1985, 236, 289-296. (21) Wolfe, J.; Bryant, G. Cryobiology 1999, 39, 103-129. (22) Hempel, E.; Hempel, G.; Hensel, A.; Schick, C.; Donth, E. J. Phys. Chem. B 2000, 104, 2460-2466.
